A versatile system for fast screening and isolation of cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting.

BACKGROUND

In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.

RESULTS

Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase promoter in , which allowed us to rapidly isolate cellulase hyperproducers from progenies transformed with a dedicated transcriptional activator and from an atmospheric and room temperature plasma-created mutant library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing and ) affecting regulatory stages beyond transcription. Using this method, cellulase hyperproducers were also successfully isolated from an -mediated random insertional mutant library.

CONCLUSIONS

The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.